title: Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies.
abstract: High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4M urea. The activity of rGST was assayed in 2M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies.
authors.affiliation: Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia; Friedrich-Alexander-Universität Erlangen-Nürnberg, Egerlandstraße 1, Erlangen, Germany.
authors.forename: Igor S
authors.initials: IS
authors.lastname: Mačinković
authors.forename: Mohamed
authors.initials: M
authors.lastname: Abughren
authors.forename: Ivan
authors.initials: I
authors.lastname: Mrkic
authors.forename: Milica M
authors.initials: MM
authors.lastname: Grozdanović
authors.forename: Radivoje
authors.initials: R
authors.lastname: Prodanović
authors.forename: Marija
authors.initials: M
authors.lastname: Gavrović-Jankulović
mesh_terms: D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D003124:Colorimetry; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D002479:Inclusion Bodies / Q000201:enzymology*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D014508:Urea / Q000737:chemistry / Q000378:metabolism
chemical_list: D000485:Allergens; D011993:Recombinant Fusion Proteins; D014508:Urea; D005982:Glutathione Transferase
keywords: GST; GST-tagged proteins; Inclusion bodies; Urea